Organophosphorous compounds (OPs) act as potent inhibitors of acetylcholine-hydrolyzing enzymes. Their ability to inhibit acetylcholinesterase, the primary synaptic regulator of cholinergic transmission, can result in highly toxic effects to humans ranging from irreversible neurological damage to death. OPs can be found in several forms, including relatively benign insecticides and far more troublesome weaponized chemical agents. Prior to World War II and extending into present times, various forms of OPs have been developed and deployed as nerve agents for nefarious purposes. These agents are classified into two general groups: 1) G agents, including Tabun (GA), Sarin (GB), Soman (GD) and Cyclosarin (GF); and 2) V agent, VX. G agents are generally non-persistent, volatile liquids in contrast with the highly persistent, non-volatile and more active VX compound. In spite of broad agreement to ban and destroy these chemical weapons, the relative ease of their synthesis and deployment makes these agents ideal tools for terrorist activities bringing about high risk to both civilian and military populations. Deployment of these agents poises an immediate health risk through respiratory or skin exposure as well as a latent threat through persistent residues on solid surfaces requiring extensive operational decontamination before reuse.
As an example, the release of sarin gas in the Tokyo subway system in 1995 provides an unfortunate illustration of the vulnerability of large population centers to chemical weapons of mass destruction (WMDs) and their potentially devastating effects. Within such a confined location, many individuals perished upon exposure to such an OPs agent. With such possible threats looming around the world (the current situation in Syria is yet another incidence), there exists the urgent need for strategies to address both human health risks as well as operational decontamination.
Acute exposure to organophosphate nerve agents is typically treated through repeated dosing of a cocktail of atropine, oxime reactivators (including 2-pyridine aldoxime methyl chloride, or 2-PAM) and anti-convulsants. These treatments suffer from significant adverse reactions, difficult compliance, and inadequate efficacy. New strategies to improve the performance of current drugs, reduce their dosage, or increase efficacy through added modes of action are required. Butyrylcholinesterase (BuChE) is a major serum human cholinesterase that shows remarkable promiscuity with regards to the chemical substrates that it binds and hydrolyzes. This property allows it to readily bind environmentally occurring OPs (pesticides) as well as both G and V classes of chemical nerve agents. In spite of a high binding constant (Km) to these diverse chemical agents, the hydrolysis (Kcat) is very slow leading to a functional inhibition stoichiometry of 1 enzyme:1 OP molecule. Many groups have diligently sought to identify enzymatically active agents of both human and bacterial origin. As such, investigations have focused greatly on BuChE developments.
There are other conditions and situations within mammalian subjects, at least, wherein BuChE has proven to be of significance. For instance, it has been realized that certain neurological conditions, such as Alzheimer's, have shown a reliance upon butyrylcholinesterase for proper function and reduction in possible neural degradation. Likewise, certain physiological conditions caused by addictive substances, cocaine, for example, may be treated through the introduction of BuChE as a treatment. There are also enzyme replacement therapies including the utilization of introduced butyrylcholinesterase to overcome subject deficiencies in natural amounts present within a body or even through the presence of mutated BuChE enzymes genes that cause reduced amounts to be generated within a subject organism. In essence, even though the potential for OPs intoxication treatments are of significance in the utilization of BuChE of any type, there exist other situations wherein a need for effective BuChE production can be of great benefit, as well.
Early studies demonstrated that hBuChE purified from the sera of equine and human sources could protect mice, guinea pigs and non-human primates from 3-5 LD50 doses of various OP nerve agents of both classes. The human serum derived BuChE shows tetrameric nature and glycan structures terminated with sialic acid leading to about a 73 hour half-life in the serum of experimental animals, thereby providing an enzyme that has been shown to be safe in human clinical trials. However, the requirement of 200 mg of the enzyme to treat a dose of 2-5 LD50 of soman (in an average sized human) renders sera sourcing unfeasible due to volumes of source material available and low yields. Recombinant BuChE (rBuChE) has been produced from transgenic goats but shows primarily monomer or dimer structure, little sialic acid termini and a rapid half-life in serum. Modification with polyethylene glycol is required to achieve a favorable pharmacokinetic profile, and has yet to be properly tested for human safety through clinical trials. Additionally, both sources of BuChE, expired human plasma or transgenic goats, appear inadequate in amount and too high in cost to provide the needed amounts of BuChE for at-risk military and civilian populations.
Because of the promise of potency, specificity, and safety profile, rBuChE is an appealing platform for OP protection. However, addressing the challenges of weapons of mass destruction (WMD) with enzymatic products presents several unique manufacturing challenges. Scale is important to address the various applications related to WMD protection and response. Modest level production can be used to provide both preventative and post-exposure responses for military populations in at-risk areas. Extremely high levels of production will be necessary to insure adequate supplies of product to address civilian exposure in the case of WMD release, as well. Typical production methods for monoclonal antibodies (mAbs) use mammalian cell reactors. Whereas this approach has been successfully used to address diseases with predictable supply requirements, large-scale mammalian culture is not well suited for rapid response and varying scale production due to capital requirements associated with cell growth facilities. Space and use amortization does not provide a conducive incentive for the enormous costs (e.g., in excess of $500M, from some estimates) required to build a suitable upstream facility. Additionally, the timeframes for needed product turnaround cycles are generally inadequate for continuous supply options. Furthermore, as new enzymatic products become available to address broader chemical structures and specificities, the process from construct development to cGMP production can reach 2-3 years in duration due to cell line optimization, process adaptation and requisite scale-up requirements (particularly in terms of mammalian-based production sources)(not to mention the general costs with husbandry, sanitation, feeding, etc., for such animals, including new generations thereof). Finally, traditionally manufactured mAbs (e.g. CHO or NS0 cells) have insufficient sialylation and other glycan modification capabilities to potently provide a long-lasting protective and therapeutic product necessary for the unpredictability of nerve agent exposure. Mammalian cell lines that offer minimal sialylation or chemical sialylation methods are unpredictable and are, among other things, subject to high royalty rates stacking onto the already significant production costs. Such escalated cost structures thus disfavor their consideration as a solution for WMD challenges. These limitations indicate a distinct desire for a new, more scalable, responsive and efficacious production strategy for such an enzyme product.
Of further importance is that previous work on BuChE structures could not provide a tetramer formation coupled with sialylation results. In particularly, it was determined that sialylation of cells could be accomplished for recombinant butyrylcholinesterase products, but the ability to provide tetramers thereof were impossible, particularly within mammalian cell bases. Thus, even though a butyrylcholinesterase platform is quite attractive for a number of treatment purposes, particularly within mammalian systems, the ability to produce not only cost-effective products in this manner, but also such products that exhibit suitable compatibility for mammalian treatments (e.g., sialylated and tetramerized), have yet to be developed. To date, in essence, there simply have not been any effective developments that provide reliable sources of BuChE with sialylated tetramer formations through repeatable processes and at low overall costs in comparison.